Am J Physiol Lung Cell Mol Physiol 308: L287–L300, 2015. First published December 5, 2014; doi:10.1152/ajplung.00229.2014.

Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension Sukrutha Chettimada,1 Rakhee Gupte,1 Dhwajbahadur Rawat,1 Sarah A. Gebb,3 Ivan F. McMurtry,2,4,5 and Sachin A. Gupte1,5 1

Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, Alabama; Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, Alabama; 3Department of Cell Biology and Neurosciences, College of Medicine, University of South Alabama, Mobile, Alabama; 4Department of Medicine, College of Medicine, University of South Alabama, Mobile, Alabama; and 5Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, Alabama 2

Submitted 18 August 2014; accepted in final form 1 December 2014

Chettimada S, Gupte R, Rawat D, Gebb SA, McMurtry IF, Gupte SA. Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 308: L287–L300, 2015. First published December 5, 2014; doi:10.1152/ajplung.00229.2014.—Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxiainducible factor 1␣ (HIF-1␣), which directs the cells to synthesize less contractile (myocardin and SM22␣) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1␣, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxiainduced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension. smooth muscle phenotype; NADPH; cell cycle; HIF-1␣; KLF; myocardin; reactive oxygen species; redox; SM22␣; Sp1

(PH) is a major cause of morbidity and mortality in patients with several different clinical conditions, and the incidence of PH is increasing around the world. The pathophysiology of PH is heterogeneous. Current medical treatment is inadequate to reverse the complex mechanisms

PULMONARY HYPERTENSION

Address for reprint requests and other correspondence: S. A. Gupte, Dept. of Pharmacology and Center for Pulmonary Hypertension, New York Medical College, 15 Dana Rd., Valhalla, NY 10595 (e-mail: [email protected]). http://www.ajplung.org

responsible for the progressive increases in pulmonary vascular resistance, and severe PH remains debilitating and deadly. Moderate hypoxia-induced PH is associated with medial thickening of muscular pulmonary arteries (PAs) and neomuscularization of arterioles. This is believed to occur by transient proliferation and subsequent hypertrophy of the medial PASM cells (43). Severe PH, such as that in idiopathic and heritable pulmonary arterial hypertension (PAH), is associated with the additional formation in distal PAs of complex cellular and fibrotic neointimal and plexiform lesions that involve the proliferation of both PASM and endothelial cells (31, 43). Thus aberrant cell proliferation and decreased apoptosis apparently occur in the vessel wall during the development of PH and PAH (47). However, the mechanisms underlying this aberrant cell growth in most forms of PH remain unclear. Glucose is essential to sustain life and is useful for many transactions in the cell. The products or by-products generated from catabolism of glucose are used by many chemical reactions that control cell survival and death. For example, glucose6-phosphate oxidized in the glycolytic pathway generates e⫺ donors that reduce molecular oxygen to generate energy in the mitochondrial respiratory chain. Also, when the cell has surplus energy, glucose-6-phosphate is shunted to the pentose phosphate pathway wherein the cell produces both NADPH that is required to protect the cell from oxidative damage and ribose sugars that are required for de novo synthesis of RNA and DNA. Although studies have linked metabolism to etiology of PH (4, 14, 26, 46), there is little known about the role of cellular metabolism in the pathogenesis of PA remodeling in the various forms of PH (15). Thus study of the links between metabolic adaptation and pulmonary vascular diseases would be useful to gain insight into the roles of metabolism in the pathogenesis of PH and PAH. In perfused lungs and isolated PAs, glucose-6-phosphate dehydrogenase (G6PD) activity is increased by hypoxia, and the G6PD overactivation has a temporal relationship with hypoxic contraction of PAs (9, 18, 20, 21). More recently, we have demonstrated that contractile protein [SM22␣ and smooth muscle myosin heavy chain (SM-MHC)] expression is markedly decreased in PAs exposed to hypoxia for 12 h in vitro. Interestingly, pretreating the arteries with G6PD inhibitors prevents the decreased expression of contractile proteins via protein kinase G-dependent pathway (9). From these observations, we predict that the hypoxia-induced increase in G6PD

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activity probably plays a critical role in changing/switching PASM cell phenotype and eliciting PASM cell proliferation during the development of PH/PAH. Therefore, this study was undertaken to elucidate the molecular mechanisms associated with the metabolic adaptation-induced phenotypic changes. Namely, we sought to determine the mechanisms through which increased G6PD downregulates myocardin, a cotranscription factor that controls contractile protein expression and promotes cell cycle. This study was performed in both a cell culture system and in vivo chronic hypoxia-induced PH and Sugen 5416 (SU)/hypoxia/normoxia-induced PAH rat models. Here we demonstrate that G6PD overactivation played a novel role in switching smooth muscle phenotype by increasing Sp1 and hypoxia-inducible factor 1␣ (HIF-1␣), which control the cell cycle, and decreasing myocardin via Sp1 and HIF-1␣ in PASM cells exposed to hypoxia in vitro and in PAs of PH and PAH rats. MATERIALS AND METHODS

Cell Culture

GUCGUACACUUtt) that specifically and efficaciously downregulated G6PD (based on our preliminary results) and a scramble sequence (negative control) were custom cloned in an adenoviral vector under H1 promoter by GeneScript to drive the short hairpin (sh) RNA expression. The adenoviral vector also carries a GFP marker (coral GPF) under CMV promoter control, which is a useful marker to estimate efficiency of transfection. These vector-based shRNAs were packaged in adenoviruses by Welgen Laboratories. Adenoviral stock [3 ⫻ 1013 plaque-forming units (pfu)], containing these two shRNAs (G6PD and scramble), was diluted by threefold (1012 pfu) and used for transfecting cultured cells. Glucose Uptake Assay Glucose uptake was measured in normoxia and hypoxia. 6-[N(7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-6-deoxyglucose (6-NBDG), a nonhydrolyzable fluorescent-tagged glucose analog was added to PASM cells in 96-well plates at a 500-␮M final concentration. After incubating for 72 h, wells were rinsed with 1⫻ PBS and fluorescence was measured at 470/520 nm in a Flx800 microplate fluorescence detector (BioTek Instruments) and confirmed by immunofluorescence microscopy. Pyruvate and Lactate Assays

Pulmonary artery smooth muscle cells. Pulmonary artery smooth muscle (PASM) cells were isolated from intra-lobar, second order rat PAs (Cell Core, Center for Lung Biology, University of South Alabama). These cells were cultured for a week, and then several clones were isolated. Spindle-shaped PASM cells were identified and probed for smooth muscle protein markers. Only those cells (passages 2–7) that expressed smooth muscle markers were used for this study. PASM cells were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY) for a span of 48 h and then used for further experiments.

Protein was extracted from cells and pyruvate and lactate assays were performed using corresponding kits from Biovision (Milpitas, CA). Briefly, the reaction mixture containing assay buffer, colorimetric probe, and enzyme was added to each well in a 96-well plate containing equal volume of protein extract. After incubation in the dark for 30 min, absorbance was measured at 570 nm in a microplate reader. A standard curve was plotted alongside using lactate/pyruvate provided in the kit. The amount of lactate or pyruvate in the samples was determined using corresponding standard curves.

Hypoxia Treatment

Superoxide Assay

After expansion, PASM cells were incubated in hypoxic chamber (InvivO2 300; Ruskin Technology) for different time periods, maintaining different O2 levels (from 3 to 80% as explained in each experiment) and 5% CO2 levels.

Lucigenin chemiluminescence. Protein extracted from PASM cells was treated with lucigenin (5 ␮mol/l) in black 96-well plates, and chemiluminescence was measured in a microplate reader (BioTek Instruments). Readings were normalized to protein content of corresponding sample.

Western Blot Analysis

Dihydroethidine Staining

Protein was extracted from cells using NP-40 lysis buffer (50 mmol/l Tris·HCl pH 7.4, 150 mmol/l NaCl, 0.5% NP-40, 100 mmol/l PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 200 mmol/l pepstatin). Thirty-five micrograms of sample were loaded and run on SDS-PAGE gels, transferred to nitrocellulose membranes, and subsequently exposed to primary and secondary antibodies and detected by ECL on autoradiography film. G6PD Activity

Hydroethidine, an oxidative fluorescent dye, was used to localize superoxide production in PASM cells. In brief, after incubation in normoxia and hypoxia with/without G6PD inhibitors, dehydroepiandrosterone (DHEA)/6-aminonicotinamide (6AN) for 72 h, PASM cells were incubated with hydroethidine (10 ␮mol/l; at 37°C for 60 min). Fluorescent images were captured at ⫻40 magnification using Zeiss microscope and analyzed using ImageJ software. Bromodeoxyuridine Assay

G6PD activity was measured in the protein extracts by estimating the reduction of NADP⫹ to NADPH. NADPH fluorescence was detected at 340 nm (Ex) and 460 nm (Em) using an Flx800 microplate fluorescence detector (BioTek Instruments, Winooski, VT).

siRNA. PASM cells cultured in a 12-well plate were transfected for 72 h with 100 nmol/l siGENOME SMARTpool siRNA targeting G6PD (Ambion, Austin, TX) using Fugene 6 transfection reagent (Promega, Madison, WI). Control experiments were performed using a nontargeting/scrambled (NT) siRNA (negative control).

DNA synthesis was measured using the bromodeoxyuridine (BrdU) cell proliferation assay kit (Millipore, Billerica, MA). PASM cells (seeded at a density of 2,500 cells/well) cultured in 96-well plates were exposed to hypoxia or normoxia for 72 h. BrdU was added 24 h before end of hypoxic/normoxic incubation. Cells were then fixed and incubated with mouse anti-BrdU antibody followed by peroxidase-conjugated anti-mouse antibody incubation. Color was developed by addition of 3,3=,5,5=-tetramethylbenzidine peroxidase substrate. After a 30-min incubation, the reaction was stopped with stop solution and BrdU intensity was measured colorimetrically at 450 nm.

Short Hairpin RNA

Cell Number Assay

We developed adenoviral vectors to deliver siRNA into cultured cells. Briefly, the G6PD-specific siRNA gene sequence (CGGAAAC-

Cell numbers were measured by CyQuant cell proliferation assay kit (Life Technologies) as per manufacturer’s instructions. Briefly,

Silencing of G6PD

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PASM cells were seeded at a density of 2,500 cells/well in 96-well plates. After 48 h, plates were incubated in normoxia or hypoxia for an acute (2 and 8 h) or prolonged (48 and 72 h) period. Following incubation, well plates were frozen at ⫺80°C overnight. Lysis buffer and fluorescent-DNA-binding dye mixture (provided in kit) were added to each well and incubated for 5 min in dark. Plates were read at 480/520 nm. A standard curve was plotted alongside using PASM cells. Cell number was determined using standard curve. Cell Cycle Analysis Cell cycle analysis was done by flow cytometric method using propidium iodide dye. Cells were trypsinized and rinsed with 1⫻ PBS followed by fixing with 70% ethanol overnight at 4°C. Fixed cells were rinsed with 1⫻ PBS and incubated in 1 ml propidium iodide solution (0.01 mg/ml propidium iodide, 0.1 mg/ml RNAseA, and 0.1% Triton X-100 in 1⫻ PBS). Stained cells were passed through 40-␮m cell strainer and analyzed in BD FACSCanto II flow cytometer. Histograms were analyzed using Modfit LT software program. Quantitative Real-Time RT-PCR Total RNA was extracted from PASM cells using miRNeasy kit (no. 217004; Qiagen, Valencia, CA). Quantitative real-time RT-PCR was performed using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad, Hercules CA). All Ct values were normalized to LDH-A Ct of corresponding sample. Samples were run on 4% agarose gel to confirm amplification of a specific product. Immunoprecipitation One milligrams of protein extract was precleared with agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Pull down was done with anti-G6PD antibody (Sigma-Aldrich, St Louis, MO) and agarose beads overnight at 4°C. The beads-antibody-protein complex was washed and resuspended in 2⫻ SDS buffer and run on SDS-PA gel. Immunoblotting was performed with anti-ubiquitin antibody (Cell Signaling, Danvers MA). Immunoprecipitation and Liquid Chromatography-Tandem Mass Spectrometry G6PD protein was pulled down using anti-G6PD antibody (Santa Cruz Biotechnology) immobilized on agarose beads. Following rotation at 4°C overnight, the beads were washed with lysis buffer, and the protein was eluted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS; Applied Biomics, Hayward, CA). NanoLC fractionation and matrix-assisted laser desorption ionizationtime of flight/time of flight (MALDI-TOF/TOF) were followed by a standard search of the National Center for Biotechnology Information and SwissProt databases using MASCOT. Immunocytochemistry Cells cultured on glass coverslips were rinsed with 1⫻ PBS and fixed with fixing solution (3.7% paraformaldehyde and 0.2% Triton X-100) followed by blocking with 0.5% BSA. Primary antibody incubation was done overnight at 4°C after which samples were incubated with secondary antibody (anti-mouse Alexa Fluor 488 or Alexa Flour 568 and anti-rabbit Alexa Fluor 568 or Alexa Fluor 488; Life Technologies) for 1 h at room temperature. The nucleus was stained with DAPI (1 ␮g/ml) and coverslips were mounted using DAKO mounting medium (DAKO, Carpinteria, CA). Imaging was done on Nikon-A1 confocal microscope.

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ing the rats to hypoxia (10% O2) for 3 wk in normobaric chambers. PAH was induced as follows: rats were injected subcutaneously with SU (20 mg/kg) and exposed to normobaric hypoxia (10% O2) for 3 wk. They were returned to normoxia (21% O2) for an additional 10 –11 wk (total 13–14 wk after SU injection). PH and PAH rats (5 from each group) were used for histological examination. An additional five rats from PH and PAH groups were treated with DHEA (1% daily diet) for 3 (PH) or 5 (PAH) wk and then used for histological examination. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of South Alabama. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Immunofluorescent Staining Paraffin-embedded lung sections (5 ␮m) of normoxic and 5-wk hypoxic rats were deparaffinized and heated with 1⫻ citrate buffer. Endogenous peroxidase activity was suppressed with 3% H2O2 treatment. Sections were blocked with blocking serum (Vectastain Universal Elite ABC kit; Vector Laboratories, Burlingame, CA). Slides were incubated with primary antibodies over night at 4°C. Secondary antibody incubation was done at room temperature for 1 h followed by avidin-biotin complex incubation for 30 min and developed with diaminobenzidine. Nucleus was stained with hematoxylin. For immunofluorescence staining, slides were incubated with Alexa Fluorconjugated secondary antibodies (Life Technologies) for 1 h at room temperature. The nucleus was stained with DAPI (1 ␮g/ml). Imaging was done on Nikon-A1 confocal microscope. Cloning of Human Myocardin Promoter and Dual Luciferase Promoter Assays Human myocardin promoter sequence was obtained from the Cold Spring Harbor Laboratory’s TRED promoter database. The 715-bp sequence was used to amplify the human Myocardin promoter. Briefly, 2 ␮g of the genomic DNA isolated from human airway smooth muscle cells were used as template to amplify the 715-bp human myocardin promoter region using a high expand fidelity PCR kit from Roche in a 50-␮l reaction. The amplified PCR product was analyzed on 0.8% agarose gel and subcloned into the TA original TOPO vector between Nhe I and Hind III restriction sites followed by ligation into the pGL-Basic vector. HEK-293T/17 cells (1 ⫻ 105) were cotransfected with the human myocardin promoter plasmid with a firefly luciferase reporter (1 ␮g/well) and a Renilla luciferase control vector pRL-SV40 (0.1 ␮g/well; Promega) using 2 ␮g of FUGENE 6 for 24 h under normoxic and hypoxic conditions, respectively, or by coexpressing the human myocardin promoter plasmid with a firefly luciferase reporter (1 ␮g/well), G6PD cDNA (5 ␮g/well), and a Renilla luciferase control vector pRL-SV40 (0.1 ␮g/well) for 24 h under normoxia. The activities of the firefly and Renilla luciferase were measured using the Dual Luciferase Reporter Assay System from Promega. Luminescence was measured using a Synergy2, Biotek luminometer. Statistical Analysis Statistical analyses were performed using GraphPad Prism 5 software. Values are presented as means ⫾ SE. Data were analyzed using ANOVA, and post hoc analysis was done by Bonferroni test. Differences between two groups were analyzed by Student’s t-tests. Values of P ⬍ 0.05 were considered significant. RESULTS

G6PD Activity and Expression Are Increased in PASM Cells by Hypoxia

Hypoxia-Induced PH and PAH Adult male Sprague-Dawley rats weighing 180 –220 g (Harlan Laboratories, Indianapolis, IN) were made pulmonary hypertensive by two established methods. Briefly, hypoxic PH was induced by expos-

The PASM cells cultured in 21% O2 were first analyzed for the contractile markers ␣-actin, calponin, tropomyosin,

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SM-MHC, myosin light chain (MLC), SM22␣, and myocardin by immunofluorescence staining (Fig. 1A). These cells expressed ␣-actin, calponin, tropomyosin, SM-MHC, MLC, SM22␣, and myocardin. PASM cells were then exposed to different O2 concentrations, and G6PD expression and activity were determined. As demonstrated in Fig. 1B, G6PD

expression and activity decreased with increasing (3– 80%) concentration of O2. To characterize the time course of changes in G6PD expression and activity in response to hypoxia and to determine whether this response was unique to PASM cells, coronary artery smooth muscle and PASM cells were exposed to either

Fig. 1. Pulmonary artery smooth muscle (PASM) cells isolated from second order rat pulmonary arteries express contractile proteins, and glucose-6-phosphate dehydrogenase (G6PD) is O2 sensitive in PASM cells. A: images showing PASM cell morphology [bright field (BF)] and staining for contractile: SM22␣, tropomyosin, smooth muscle (SM) ␣-actin and calponin (all green) and myocardin (Myocd) and SM-myosin heavy chain (MHC; all red). MLC, myosin light chain. B: G6PD protein (top) and activity (bottom) decreased with increasing O2 tension. IB, immunoblot. C–E: G6PD protein expression is increased by acute and prolonged hypoxia in PASM but not in coronary artery smooth muscle (CASM) cells (top). G6PD activity is increased in PASM cells by acute and prolonged hypoxia (bottom). F–G: exposure of PASM cells to hypoxia for 72 h increased glucose uptake {as indicated by 6-[N-(7-nitrobenz-2oxa-1,3-diazol-4-yl]amino)-6-deoxyglucose (6NBDG) uptake; top} and lactate-to-pyruvate ratio (bottom); n ⫽ 5 experiments done in triplicate. Gray and black bars represent normoxia (Nx) and hypoxia (Hx) groups, respectively.

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21 or 3% O2 for 2, 8, 48, and 72 h. G6PD activity and expression increased within 2 h of exposure to hypoxia and remained elevated for up to 72 h exclusively in PASM cells (Fig. 1, C–E). At 72 h of exposure of the PASM cells to hypoxia, glucose uptake (as indicated by 6-NBDG) as well as the lactate-topyruvate ratio was increased (Fig. 1, F and G). G6PD Translation Is Upregulated in Hypoxic PASM Cells To determine how G6PD expression was induced by hypoxia, we measured G6PD mRNA levels in PASM cells. There were no significant changes in G6PD transcript levels at any time of hypoxic exposure (Fig. 2A). We also confirmed these results by normalizing G6PD-mRNA by GAPDH-mRNA (normoxia: 1.17 and hypoxia: 1.19). These results suggested that G6PD expression was regulated at a posttranscriptional level. To determine whether acute hypoxic exposure increased G6PD translation, cells were incubated either with cycloheximide (30 ␮g/ml) or at 4°C for 2 h under normoxic and hypoxic conditions. Interestingly, both cycloheximide and low temperature suppressed the hypoxia-induced G6PD expression, while G6PD expression was unaffected in normoxic conditions (Fig.

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2B). Since H2O2 increases G6PD activity and promotes the translation of the Nrf2 protein, which is known to increase antioxidant enzyme expression (36), we further sought to determine whether increased H2O2 in hypoxic PASM cells induced the G6PD translation. Therefore, we treated PASM cells with the H2O2 scavenger, pegalated (peg)-catalase (100 U/ml), and assessed G6PD expression. We found that pegcatalase suppressed G6PD expression at 2 h (Fig. 2B) but not at 72 h (data not shown) of hypoxia. Next, to determine whether G6PD expression was downregulated by proteasomal degradation in normoxia, PASM cells were treated with the proteasome inhibitors lactacystin (5–10 ␮mol/l) and MG132 (20 –100 ␮mol/l). G6PD expression increased with increasing doses of proteasomal inhibitors (Fig. 2C). Also, pull-down assays with G6PD antibody stained positive for ubiquitin upon immunoblotting (Fig. 2D). Interestingly, ubiquitination of G6PD was downregulated in hypoxic compared with normoxic PASM cells (Fig. 2D). PASM Cell Proliferation Is Induced by Increased G6PD Activity in Hypoxia To determine the role of G6PD in PASM cell proliferation, PASM cells were first exposed to prolonged hypoxia and treated with the G6PD inhibitors 6AN or DHEA for 72 h. Both 6AN and DHEA decreased activity without affecting expression of G6PD under normoxic and hypoxic conditions (Fig. 3, A and B). Next, PASM cell numbers were measured after 2-, 8-, 48-, and 72-h exposure to hypoxia. Cell numbers increased at 48 and 72 h of hypoxia (Fig. 3C). To test whether G6PD played a role in the hypoxia-induced increase of PASM cell numbers, we examined the effect of 6AN and DHEA on cell numbers. Cells were grown for 72 h in either normoxia or hypoxia, and then treated with G6PD inhibitors for an additional 48 h. As demonstrated in Fig. 3D G6PD inhibitors stalled PASM cell growth. To further confirm the role of G6PD in the hypoxia-induced increase of PASM cell numbers, cells were treated with G6PDspecific siRNA. G6PD-siRNA decreased G6PD expression as well as PASM cell numbers compared with nontargeting siRNA controls (Fig. 3, E and F). Interestingly, ectopic overexpression of G6PD using adenoviral transfection (Fig. 3G) increased PASM cell numbers under normoxic conditions compared with vector control (Fig. 3H). Expression of Contractile Protein in PASM Cells Is Reduced by Increased G6PD Activity in Hypoxia

Fig. 2. G6PD translation is upregulated by hypoxia in PASM cells. A: G6PD mRNA levels did not increase with acute or chronic hypoxia. B: inhibition of translation by cycloheximide treatment (30 ␮g/ml) or by incubating cells at 2– 4°C and peg-catalase (100 U/ml) suppressed the increase in hypoxiainduced G6PD expression. (*P ⬍ 0.05 vs. normoxia; #P ⬍ 0.05 vs. hypoxia control). C: treatment with proteasome inhibitors: MG132 (20 and 100 ␮mol/l) and lactacystin (5 and 10 ␮mol/l) increased G6PD expression. D: PASM cells were treated with proteasome inhibitors: MG132 and lactacystin followed by pull down with anti-G6PD antibody and immunoblotting with anti-ubiquitin antibody. G6PD forms complex with ubiquitin suggesting that G6PD expression is also regulated by ubiquitin-proteasome-mediated degradation; n ⫽ 5 experiments done in triplicate.

To test the hypothesis that hypoxia makes the PASM cells less contractile and more proliferative and that G6PD plays a critical role in this switching of phenotype, we exposed cultured PASM cells to normoxia (21% O2) or hypoxia (3% O2) for 72 h and determined the expression of contractile proteins by immunocytochemistry and immunoblotting. Immunocytochemistry data showed that expression of myocardin, tropomyosin, SM22␣, and SM-MHC decreased upon exposure to hypoxia compared with normoxia (Fig. 4A). Consistently, immunoblotting data also showed that myocardin and SM22␣ decreased (P ⬍ 0.05) in PASM cells exposed to hypoxia compared with normoxia (Fig. 4B). The hypoxia-induced decreases in myocardin and SM22␣ expression were prevented

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Fig. 3. PASM cell numbers are increased by prolonged exposure to hypoxia. A: 6-aminonicotinamide (6AN; 1 mmol/l) and dehydroepiandrosterone (DHEA; 100 ␮mol/l) treatment did not affect G6PD protein expression in normoxia or hypoxia (72 h; G6PD normalized to actin expression). B: 6AN and DHEA treatment decreased G6PD activity in normoxia and hypoxia (72 h; *P ⬍ 0.05 vs. untreated control; #P ⬍ 0.05 vs. normoxia control). C: PASM cell numbers increased with prolonged hypoxia (48 and 72 h) but not with acute hypoxia (2 and 8 h; *P ⬍ 0.05 vs. time-matched normoxia control). D: cells were grown for 72 h in either normoxia or hypoxia and, then, treated with G6PD inhibitors for an additional 48 h, which inhibited PASM cell growth (*P ⬍ 0.05 vs. untreated control; #P ⬍ 0.05 vs. normoxia control). E and F: treatment with G6PD-siRNA significantly decreased G6PD expression and PASM cell numbers in normoxia and hypoxia [*P ⬍ 0.05 vs. nontargeting (NT) control; #P ⬍ 0.05 vs. hypoxia NT control]. G and H: G6PD overexpressed in PASM cells either with adenoviral transfection of GFP (vector) or GFP tagged-G6PD (Ad-G6PD) increased cell numbers in normoxia (*P ⬍ 0.05 vs. vector control); n ⫽ 5 experiments done in triplicate.

by inhibition of G6PD with 6AN, DHEA, and siRNA. In contrast, ectopic overexpression of G6PD decreased myocardin expression in normoxic PASM cells. Cell Cycle and Cell Cycle Protein Expression Are Upregulated by Increased G6PD Activity in Hypoxia We also investigated whether hypoxia-induced G6PD activation alters the cell cycle of PASM cells. Cell cycle analysis was performed by flow cytometry. G6PD inhibition with DHEA or 6AN decreased the number of hypoxic cells in S and G2/M phase of cell cycle (Fig. 4C). While cyclin D1 (the protein essential for G1/S transition) expression decreased in hypoxic PASM cells and was further reduced by 6AN and

DHEA (Fig. 4D), expression of cyclin A (the protein required for S-phase progression) increased in hypoxic PASM cells and was suppressed by G6PD inhibitors (Fig. 4E). This finding was further confirmed by a BrdU assay, which showed that hypoxiainduced increase in DNA synthesis was suppressed by 6AN and DHEA (Fig. 4F). Phospho-histone H3 (essential for cytokinesis) is often used as mitotic index marker. Expression of phospho-histone H3 increased in hypoxic PASM cells, and this increase was suppressed by 6AN and DHEA treatment (Fig. 4G). The role of G6PD in promoting expression of proliferative proteins was further confirmed by G6PD knockdown; G6PD-siRNA delivery to PASM cells decreased phospho-histone H3 (Fig. 4H).

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Fig. 4. Phenotypic changes in hypoxic PASM cells. A: immunofluorescence staining demonstrates expression of contractile markers in normoxic and hypoxic PASM cells. B: immunoblot analysis of PASM cells exposed to prolonged hypoxia (72 h) shows decreased expression of myocardin and SM22␣ proteins (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control). G6PD inhibition with siRNA and overexpression increased and decreased myocardin expression, respectively. C: cell cycle analysis of PASM cells showing an increase in the S and G2/M phase in hypoxia. D and E: expression of cell cycle proteins, cyclin D1, and cyclin A in PASM cells exposed to hypoxia are shown (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control). F: bromodeoxyuridine (BrdU) assay showing an increase in DNA synthesis in hypoxic PASM cells (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control). G: hypoxia-induced increase in phospho-histone H3 (H3P). G6PD inhibition with DHEA (100 ␮mol/l) or 6AN (1 mmol/l) normalized H3P levels (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control). H: G6PD inhibition with siRNA decreased phospho-histone H3 expression. I: in contrast, overexpression of G6PD increased phospho-histone H3. (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control).

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Conversely, ectopic overexpression of G6PD increased phospho-histone H3 levels (Fig. 4I). H2O2 Does Not Play a Major Role in G6PD-Mediated Increase of Myocardin or Decrease Cyclin D1 Expression in Hypoxic PASM Cell Exposure of PASM cells to hypoxia for 72 h increased reactive oxygen species (ROS) levels, as determined by DHE staining (Fig. 5A) and lucigenin chemiluminescence measurements (Fig. 5B). To determine whether the increase in H2O2 under hypoxia played a role in regulating myocardin and cyclin D1 expression in hypoxic PASM cell, we treated cells with peg-catalase (100 U/ml), which scavenges ROS in PAs (19). Myocardin (Fig. 5C) and cyclin D1 (Fig. 5D) protein expression in PASM cells treated with 6AN was unaffected by peg-catalase. Expression of Transcription Factors That Control Smooth Muscle Phenotype and Cell Cycle Protein Expression Is Regulated by G6PD Myocardin protein (Fig. 4B) and mRNA (Fig. 6A) decreased in PASM cells exposed to hypoxia. We have also confirmed these results by normalizing myocardin-mRNA by GAPDH-

mRNA. Furthermore, hypoxia and ectopic overexpression of G6PD decreased myocardin promoter activity (Fig. 6B). To determine the molecular mechanisms by which G6PD downregulates myocardin, a cotranscription activator of contractile protein genes (50), and upregulates expression of proteins involved in cell proliferation, we estimated the levels of 1) Sp1, which downregulates myocardin and increases cell cycle protein expression (13, 17); and 2) HIF-1␣, which regulates expression of several cell cycle proteins (49) and elicits angiogenesis by integrating with Notch signaling (32). Interestingly, the expression of Sp1 and the nuclear-tocytosolic ratio of HIF-1␣ increased in PASM cells exposed to hypoxia for 72 h (Fig. 6, C–E). This increase was reversed by 6AN treatment, which concurrently increased myocardin (Fig. 4). Artemisinin (1 ␮mol/l), an Sp1 inhibitor that blocks binding of Sp1 to the transcription site (40), partially prevented downregulation of myocardin expression in hypoxic PASM cells (Fig. 6F). In contrast, CoCl2, which upregulates HIF-1␣ in most cell types, also decreased myocardin (by 75.5%) expression in PASM cells (Fig. 6G). Additionally, using a combination of immunoprecipitation and LC-MS/MS followed by MALDI-TOF/TOF analysis, we found an association between the G6PD and components of the Notch signaling pathway,

Fig. 5. Reactive oxygen species (ROS) do not play a role in G6PD-mediated increase in cell numbers and myocardin expression in hypoxia. A and B: DHE staining and lucigenin chemiluminescence measurements show increased ROS levels in hypoxia PASM cells. C and D: peg-catalase did not affect the 6ANinduced change in the expression of myocardin and cyclin D1.

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Fig. 6. G6PD regulates expression of myocardin-mRNA and transcription factors in normoxic and hypoxic PASM cells (*P ⬍ 0.05 vs. normoxia control). A: myocardin mRNA levels decreased in PASM cells exposed to prolonged hypoxia (72 h). B: myocardin promoter activity (V ⫹ RNx, empty luciferase vector ⫹ Renilla in normoxia; V ⫹ RHx, empty luciferase vector ⫹ Renilla in hypoxia; Myoc ⫹ RNx, myocardin promoter tagged to luciferase vector ⫹ Renilla in normoxia; Myoc ⫹ RHx, myocardin promoter tagged to luciferase vector ⫹ Renilla in hypoxia; and G6PD ⫹ Myoc ⫹ RNx, G6PD-cDNA ⫹ myocardin promoter tagged to luciferase vector ⫹ Renilla in normoxia:) determined by luciferase reporter decreased in hypoxia compared with normoxia, and by coexpressing G6PD cDNA with myocardin promoter in normoxic condition. C: representative of 5 Western blot experiments show that Sp1 and hypoxiainducible factor 1␣ (HIF-1␣) expression is increased under hypoxia in PASM cells. D and E: summary data showing G6PD inhibition with 6AN suppressed the hypoxia-induced increase in Sp1 and nuclear HIF-1␣ (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control). F: inhibition of Sp1 with artemisinin prevented downregulation of myocardin expression in hypoxic PASM cells (*P ⬍ 0.05 vs. normoxia control; #P ⬍ 0.05 vs. untreated control). G: representative immunoblot of 5 experiments showing CoCl2, an inducer of HIF-1␣, downregulates expression of myocardin in PASM cells.

which integrates with HIF-1␣ and modifies cell phenotype in arterial tissue (Table 1). Myocardin Expression Is Increased and PH Is Reduced by G6PD Inhibition To determine whether G6PD activation alters PASM cell phenotype in PAs of hypertensive lungs and contributes to the pathogenesis of PH, we performed light microscopy to determine PA remodeling and confocal microscopy to determine

myocardin and SM22␣ expression and biochemical studies to determine G6PD activity in lungs from hypoxia-induced PH and SU/hypoxic/normoxic-induced PAH rats. Light microscopy (Fig. 7A) demonstrated PA remodeling and occlusive lesions in the lungs from PAH rats. Remodeling was decreased by DHEA (1% daily diet treatment). Immnunofluorescence staining (Fig. 7B) showed that myocardin (green) expression was decreased in the medial region of PAs from hypoxia-induced PH and SU/hypoxic/normoxic-induced PAH

Table 1. LC-MS/MS with MALDI-TOF/TOF performed after G6PD pull down Top Ranked Protein Name

Accession No.

Protein MW

Protein PI

Peptide Count

Total Ion Score

Total Ion CI, %

Neurogenic locus notch homolog protein 3 ADAM metallopeptidase domain 22 Protein Wnt-4 Proto-oncogene Wnt-1 Transcription factor Sp7 Transcription factor Sp9

gi|359066825 gi|296488415 gi|359063413 gi|166795317 gi|156120931 gi|329663416

244,202 95,081 32,900 41,012 45,016 48,672

5.1 7.2 8.8 9.2 8.6 9.1

8 5 3 3 3 3

50 72 45 37 42 36

100 100 99 94 98 91

LC-MS/MS, liquid chromatography-tandem mass spectrometry; MALDI-TOF/TOF, matrix-assisted laser desorption ionization-time of flight/time of flight; G6PD, glucose-6-phosphate dehydrogenase; MW, molecular weight; PI, isoelectric point; CI, chemical ionization; ADAM, a disintegrin and metalloproteinase. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00229.2014 • www.ajplung.org

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Fig. 7. G6PD inhibition rescues myocardin and SM22␣ expression in PA of pulmonary arterial hypertension (PAH) rat and prevents hypoxia-induced pulmonary hypertension (PH). A: vascular remodeling and lesion in rat lungs from normal and PAH [Sugen 5416 (SU)/Hx/Nx]- and PAH (SU/Hx/Nx) ⫹ DHEA-treated rats. A representative of 4 experiments is shown. B: immunofluorescence staining for myocardin (green) and SM22␣ (red) in rat lungs from normal; 3-wk hypoxic and PAH- and PAH ⫹ DHEA-treated rats. Nucleus is stained with DAPI (blue). A representative of 5 experiments is shown. G6PD activity (C) and NADPH levels (D) increased in lungs of rats exposed to chronic hypoxia (5-wk hypoxia, 10% O2). DHEA treatment (1% daily diet) suppressed the hypoxia-induced increase of activity in rat lungs. E and F: regression analysis showing a positive correlation between increased G6PD and HIF-1␣ in the lungs of rat exposed to chronic hypoxia. DHEA treatment suppressed the hypoxia-induced increase in HIF-1␣. G and H: DHEA treatment suppressed the hypoxia-induced increase in mean pulmonary artery pressure and RV/LV ⫹ S ratio in PH rats.

rats. The reduction of myocardin expression in both PH and PAH models was also rescued by DHEA (1% daily diet) treatment (Fig. 7B). Additionally, G6PD activity and NADPH levels increased in the lungs of hypoxia-induced PH compared with normal rats. DHEA treatment suppressed the hypoxiainduced increase of G6PD activity and NADPH levels (Fig. 7, C and D). Interestingly, we found a positive correlation between G6PD and HIF-1␣ expression in the lungs from PH rats and DHEA reduced HIF-1␣ expression levels to those found in normal rat lungs (Fig. 7, E and F). Finally, DHEA treatment

suppressed the hypoxia-induced increase in mean pulmonary artery pressure and right ventricular hypertrophy (RV/LV ⫹ S ratio) (Fig. 7, G and H). DISCUSSION

The salient findings of this study are 1) G6PD expression and activity are increased in PASM cells by hypoxia and in lungs of PH/PAH rats, 2) increased G6PD activity and G6PDdependent redox played a pivotal role in increasing PASM cell

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proliferation by downregulating contractile protein expression in PASM cells and in PAs from PH and PAH rats and by upregulating cell cycle proteins expression in PASM cells, 3) G6PD activation decreased contractile protein and increased cell cycle protein expression by suppressing myocardin and increasing Sp1 and HIF-1␣ expression, 4) Sp1 and HIF-1␣ mediated G6PD-induced repression of myocardin expression, and 5) DHEA treatment increased myocardin and decreased HIF-1␣ expression in moderate PH and severe PAH rat arteries and lungs and decreased pulmonary arterial pressure and right ventricular hypertrophy. Therefore, our novel observations suggest that metabolic adaptation evokes PASM cell proliferation and potentially contributes to pulmonary arterial remodeling and the pathogenesis of PH and PAH. G6PD activity is increased in hypoxic compared with normoxic PASM cells. Moreover, glucose uptake is stimulated (as indicated by 6-NBDG uptake) and anaerobic glycolysis (as indicated by an increase in lactate-to-pyruvate ratio) is promoted by hypoxia in PASM cells. These results indicate that an inhibition of aerobic glycolysis/pyruvate oxidation induced by hypoxia shunted the glucose flux into the pentose phosphate pathway through activation of G6PD. In PASM and cancer cells/solid tumors, the pentose phosphate pathway activity is indeed augmented by hypoxia (16, 18, 53). Hypoxia increases glucose uptake by ⬃200% in PAs (27), inhibits aerobic glycolysis in isolated lungs (41) and PAs (18), and redirects the flux of glucose from glycolysis to the pentose phosphate pathway by inhibiting phosphofructokinase-1 activity in cancer cells (53). More surprising and interesting is our finding that hypoxia not only rapidly increased G6PD activity but also upregulated its expression by augmenting translation, without significantly changing G6PD-mRNA levels in PASM but not in coronary smooth muscle cells. Hypoxia-induced G6PD expression was inhibited by cycloheximide, which blocks the translocation of tRNA molecules and mRNA in relation to the ribosome in protein synthesis (24, 39), and by incubating the cells at 4°C, which slows all translation processes. More interestingly, the hypoxia-induced increase of G6PD was blocked by peg-catalase. This suggests that H2O2, which is well known to increase in PASM cells by hypoxia (22, 23), stimulated G6PD translation. In addition to accentuating G6PD translation, our experiments with proteasome inhibitors and coimmunoprecipitation studies also showed that G6PD is degraded via ubiquitinproteasome pathways in normoxic PASM cells and its degradation is suppressed in hypoxic PASM cells. Paradoxically, G6PD is downregulated in the systemic arteries of hyperaldosteronism-associated hypertension and type 1 diabetic patients (7, 8, 28, 42). Therefore, based on our seminal findings, we propose that G6PD expression is maintained at low levels in normoxic PASM cells by 1) a low-rate of translation and 2) a high rate of degradation and that increased hypoxia-induced H2O2 generation turns off these mechanisms acutely allowing G6PD to accumulate in PASM cells and PAs resulting in shunting of glucose flux through the pentose phosphate pathway. Metabolism is altered in PASM cells of PH and PAH animals and patients (46), but whether it is a cause or consequence of the disease is unclear. Here we demonstrate that PASM cells in S and G2/M phase were higher in hypoxia than normoxia and both inhibition and silencing of G6PD decreased hypoxia-induced proliferation of PASM cells. Moreover,

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G6PD inhibition decreased cyclin A and phospho-histone H3 expression, attenuated DNA synthesis, and arrested cell cycle in the G0/G1 phase compared with untreated hypoxic controls. In contrast, overexpression of G6PD in normoxic PASM cells increased phospho-histone H3 expression and their proliferation. This suggests that increased G6PD activity promoted PASM cell proliferation/growth in hypoxia. Likewise, elevated pentose phosphate pathway activity has been implicated to confer a selective growth advantage to cancer cells (53) and promote their growth (44) by increasing cell proliferation or reducing apoptosis that contributes to an increase in cell numbers of many cell types including cancerous cells (37). Otto Warburg postulated that inhibition of oxidative phosphorylation and activation of glycolysis, also known as the Warburg effect, was a cause of aberrant cell growth in cancer (29), and similar suggestions have been made with respect to the abnormal cell growth in PH (1, 22). It is now known that induction of pyruvate dehydrogenase kinase 1 and HIF activity in tumors is a cause of the Warburg effect (12). Additionally, it is also reported that the major function of glucose metabolism for K ras-induced, anchorage-independent cellular growth, a hallmark of transformed cells, is to support the pentose phosphate pathway to generate nucleotides and phospholipids for rapidly proliferating tumor cells (51). Interestingly, metabolism drives cell proliferation and death in which the NADPH derived from the pentose phosphate pathway plays a key role in inhibiting apoptosis (5). Changes in glucose metabolism potentially modulate several steps in cell cycle and death pathways. Glucose uptake and intermediate metabolites of glycolytic pathway control the cell cycle by influencing either expression or activity of proteins such as cyclins, cyclin-dependent kinases, and the anaphase-controlling proteins. ROS, whose levels are regulated by G6PD-derived NADPH, are also known to regulate several steps in the cell cycle (48). Elevated NADPH oxidase-derived ROS control all the cell cycle phases by influencing growth factor signaling pathways and ubiquitination of cyclins or cyclin-dependent kinases in mammalian cells (38, 48). The role of pyridine nucleotide signaling in regulating cell survival in the cardiovascular system is just emerging (33),

Fig. 8. A schematic summarizing the effect of hypoxia on G6PD and its downstream effects that contribute to smooth muscle phenotype switching from contractile to synthetic that ultimately leads to the development of pulmonary hypertension phenotype in the hypoxic rat lungs.

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and we demonstrate here that downregulation of cell cycle proteins following G6PD inhibition could be a cause of cell cycle arrest. Concomitantly, we also show that G6PD blockade by DHEA reduces remodeling of PAs in PAH rats and decreases mean pulmonary arterial pressure and right ventricular hypertrophy in PH rats. These findings imply that increased G6PD expression and activity contribute to the pathogenesis of PH and PAH. Vascular smooth muscle cells can alternate between contractile and proliferative phenotypes depending on environmental cues. In the blood vessels, smooth muscle cells mainly exist in the contractile state and express a cohort of contractile proteins that aid in maintaining the normal vascular tone of blood vessels (35). However, they switch from a differentiated (contractile) to a proliferative (synthetic) state to repair the blood vessel when the smooth muscle is injured by chemical or physical factors (35). Hypoxia reduces the promoter activity of multiple smooth muscle cell marker genes in fetal pulmonary venous smooth muscle cells and in primary PASM cells (25, 54). The phenotype of PASM cells is switched in PH as well (47). In isolated PAs, hypoxia-induced phenotypic switching, indicated by a reduction in expression of myocardin and smooth muscle contractile proteins, is prevented by G6PD inhibition (9). Consistently, our present findings suggest that G6PD inhibition prevented myocardin from decreasing in hypoxic PASM cells and in PAs of PH and PAH rats. The serum response factor (SRF)-myocardin complex upregulates the synthesis of contractile proteins, while the Klf4/Elk1 stops contractile protein gene expression by binding to SRF-myocardin complex. Consistently, SM22␣ expression decreased, which was inhibited by 6AN and DHEA, modestly in PASM cells and SM22␣ staining was reduced slightly in PAs of PAH rats, which may suggest that the SM22␣ gene may be active despite robust myocardin downregulation. It is suggested that TGF␤-SMAD and miR-145 upregulate (11, 30) and Sp1dependent activation of Klf4 (13) downregulates myocardin expression. Interestingly, G6PD-derived NADPH redox increases Sp1 binding to an oligonucleotide containing the Sp1 consensus sequence and promotes transcription of Sp1-dependent genes (3, 52). Additionally, G6PD forms a complex with other members of the Sp family, Sp7 and Sp9, of transcription factors (Table 1). Since G6PD inhibition decreased Sp1 expression and artemisinin (an Sp1 inhibitor) increased myocardin expression, we propose that G6PD-dependent upsurge of Sp1 expression and activity, at least partially, downregulated myocardin gene expression in hypoxic PASM cells and PAs. Likewise, NADPH stabilizes HIF-1␣ in human mesangial cells (34), NADPH oxidase-derived ROS activate HIF-1␣ in HepG2/H9C2 cells (2), and NADPH-dependent activation of thioredoxin reductase promotes HRE activity in human PASM cells (45). Our findings suggest that HIF-1␣ expression is increased via a G6PD-dependent manner in hypoxic PASM cells and in lungs from PH rats. Increases in the expression and activity of Sp1 and HIF-1␣ turn on the transcription of several genes, including but not limited to cyclin D, which controls G1/S transition and synthesis of S-phase proteins and other proteins that facilitate cell cycle progression (5). In contrast, HIF-1␣ is predicted to turn off myocardin activity by integrating with Notch pathway (32). We found that CoCl2, a chemical inducer of HIF-1␣, downregulated myocardin expression. We also found that G6PD associates with Wnt and Notch-3/4

(Table 1), and, hence, we propose that the NADPH redoxinduced increase of nuclear HIF-1␣ potentially integrates with Notch signaling (a redox-sensitive pathway; Ref. 10) and decreases myocardin activity in smooth muscle, which facilitates switching of PASM cell phenotype. Therefore, our novel data demonstrate that hypoxia-induced adaptive metabolic changes and increased G6PD activity played a potential role in switching the expression from contractile to proliferative proteins in hypoxic PASM cells by regulating the expression of transcription factors in a redox-dependent manner. Finally, DHEA treatment, which inhibited G6PD activity in the lungs of hypoxic rats, decreased hypoxia-induced increases in mean pulmonary artery pressure and right ventricular hypertrophy. These data show that G6PD plays a role in the development of hypoxia-induced PH and that inhibition of G6PD can relieve the symptoms of PH. Consistently, the right ventricle of PAH patients shows a significant increase in glucose uptake compared with healthy volunteers and also in these patients right ventricular glucose uptake is positively correlated with pulmonary arterial pressure (6). In summary, we have elucidated a molecular mechanism that stimulates G6PD expression and activity in hypoxic PASM cells; demonstrated that G6PD-dependent redox suppressed contractile protein expression and promoted proliferation of PASM cells, which contributes to pulmonary vascular remodeling; and provided evidence that activated G6PD plays a potential pathophysiological role in PH and PAH rats (Fig. 8). These findings support the notion that changes in metabolism in general and in glucose metabolism in particular are potentially involved in the pathogenesis of PH and, therefore, the G6PD/pentose phosphate pathway could be a new and attractive drug target for PAH therapy. ACKNOWLEDGMENTS Present address of S. Chettimada: Cancer Immunology and AIDS, DanaFarber Cancer Institute, 3 Blackfan Circle, Boston, MA 02115. Present address of R. Gupte and S. A. Gupte: Dept. of Pharmacology, New York Medical College and Center for Pulmonary Hypertension, 15 Dana Rd., Valhalla, NY 10595. GRANTS This study was support by National Heart, Lung, and Blood Institute Grant HL-085352. DISCLOSURES No conflicts of interest, financial or otherwise are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.C., R.G., I.F.M., and S.A. Gupte conception and design of research; S.C. and D.R. performed experiments; S.C., D.R., and S.A. Gupte analyzed data; S.C., R.G., S.A. Gebb, I.F.M., and S.A. Gupte interpreted results of experiments; S.C. prepared figures; S.C. drafted manuscript; S.C., R.G., S.A. Gebb, I.F.M., and S.A. Gupte edited and revised manuscript; S.C., R.G., D.R., S.A. Gebb, I.F.M., and S.A. Gupte approved final version of manuscript. REFERENCES 1. Archer SL, Gomberg-Maitland M, Maitland ML, Rich S, Garcia JG, Weir EK. Mitochondrial metabolism, redox signaling, and fusion: a mitochondria-ROS-HIF-1␣-Kv1.5 O2-sensing pathway at the intersection of pulmonary hypertension and cancer. Am J Physiol Heart Circ Physiol 294: H570 –H578, 2008. 2. Biswas S, Gupta MK, Chattopadhyay D, Mukhopadhyay CK. Insulininduced activation of hypoxia-inducible factor-1 requires generation of

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Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension.

Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertens...
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